This invention relates to novel compounds that recognize mixed sequences (i.e., GC as well as AT base pairs), and specifically bind the DNA minor groove through dimer formation.
Design and discovery of molecules that can regulate gene expression in cells in a desirable and predictable manner is a central goal of research at the interface of chemistry and biology. See, e.g., Schreiber, S. L., Bioorg. Med. Chem. 6, 1127-1152 (1998); C. Denison and T. Kodadek, Chem. Biol. 5, R129-R145 (1998); A. G. Papavassiliou, Molecular Medicine Today 358-366 (1998); R. E. Bremer, et al., Chem. Biol. 5, 119-133 (1998); J. Gottesfeld et al., Nature 387, 202-205 (1997); H. Iida, Current Opinion Biotechnology 10, 29-33 (1999). The developing field of xe2x80x9cchemical geneticsxe2x80x9d requires molecules that have the necessary selectivity to recognize target genes. See, e.g., S. Schreiber, supra, and Schreiber, S., FASEB J. 11, p.M1 (1997).
A number of aromatic diamidines have been shown to bind to the minor-groove of DNA, and to exhibit useful antimicrobial activity. Various hypotheses of the mode of antimicrobial action of the aryl amidines have been proposed. However, evidence is growing that these compounds function by complex formation with DNA and subsequent selective inhibition of DNA dependent microbial enzymes. Intervention in transcription control has been demonstrated and seems to be a plausible mode of action for structurally diverse minor groove binders. B. P. Das, et al., J. Med. Chem. 20, 531-536 (1977); D. W. Boykin, et al., J. Med. Chem. 36, 912-916 (1995); A. Kumar et al., Eur. J. Med. Chem. 31, 767-773 (1996); R. J. Lombardy, et al., J. Med. Chem. 31, 912-916 (1996); R R. Tidwell. et al., Antimicrob. Agents Chemother. 37, 1713-1716 (1993); R. R. Tidwell, R. R. and C. A. Bell, xe2x80x9cPentamidine and Related Compounds in Treatment of Pneumocystis carinii Infection,xe2x80x9d in Pneumocystis carinii, (Marcel Decker; New York, 561-583 (1993)); D. Henderson, and L. H. Hurley, Nature Med. 1, 525-527 (1995); J. Mote, Jr., et al., J. Mol. Biol. 226, 725-737 (1994); and D. W. Boykin, et al., J. Med. Chem. 41, 124-129 (1998).
Organic cations that bind in the DNA minor groove also have biological activities that range from anti-opportunistic infection to anticancer properties. See e.g., C. Bailly, in Advances in DNA Sequence-Specific Agents, Vol. 3, pp. 97-156 (L. H. Hurley, Ed. JAI Press Inc., London, UK, 1998); J. A. Mountzouris and L. H. Hurley, in Bioorganic Chemistry: Nucleic Acids, pp. 288-323, (S. M. Hecht, Ed., Oxford Univ. Press, New York, 1996); E. Hildebrant, et al., J. Euk. Microbiol. 45, 112 (1998); and K. Hopkins et al., J. Med. Chem. 41, 3872 (1998). Such compounds have provided a wealth of fundamental information about nucleic acid recognition properties, and they continue to be important models in the study of nucleic acid complexes.
The DNA minor-groove and AT sequence recognition properties of molecules of this series have been probed extensively for more than 30 years. See, e.g., C. Zimmer and U. Wahnert, Prog. Biophys. Mol. Biol. 47, 31 (1986); B. H. Geierstanger and D. E. Wemmer, Annu. Rev. Biophys. Biomol. Struct. 24, 463 (1995); W. D. Wilson, in Nucleic Acids in Chemistry and Biology, Chapter 8 (G. M. Blackburn and M. J. Gait, Eds., IRL Press, Oxford, U.K., 1996). The compound netropsin (see FIG. 1) was the first minor groove-binding compound crystallized with a B-form DNA, and the structure of the complex provided clear suggestions about the molecular basis for AT base pair sequence-specific recognition. M. L. Kopka, et al., Proc. Natl. Acad. Sci. 82, 1376 (1985). The structure of netropsin also led to the development of minor-groove binding netropsin analogs, the lexitropsins, that could specifically recognize GC base pairs and could thus have extended sequence recognition capability. See, J. W. Lown et al., Biochemistry 25, 7408 (1986); M. L. Kopka and T. A. Larsen, in Nucleic Acid Targeted Drug Design, pp. 303-374C (L. Probst and T. J. Perun, Eds., Marcel Dekker Inc., New York, 1992); and M. L. Kopka et al., Structure 5, 1033 (1997). Initial efforts in the design of such analogs did provide compounds with enhanced recognition of GC base pairs, but unfortunately, the specificity obtained was not significant. A breakthrough in this area occurred with the discovery that the monocationic compound distamycin (FIG. 1) could bind into the minor groove of some AT sequences of DNA as a stacked, antiparallel dimer. See J. G. Pelton and D. E. Wemmer, Proc. Natl. Acad. Sci. 86, 5723 (1989), and J. G. Pelton and D. E. Wemmer, J. Am. Chem. Soc. 112, 1393 (1990).
One of the early recognition principles for AT sequences was the fact that the minor groove is narrower in AT than in GC regions, and it is perhaps the most surprising feature of the dimer complex that the minor groove in B-form DNA can readily expand to the width required for dimer binding. The expansion of the groove not only allows the dimer to bind but also provides for recognition of both strands in the duplex through complementary strand recognition by the two molecules of the dimer. Replacement of pyrrole group in distamycin by imidazole provided improved GC recognition specificity with dimer complexes and current design efforts in this system have reached a high level of success. See e.g., C. L. Kielkopf, et al., Nature Struct. Biol. 5, 104 (1998); S. Whiteet al., Nature 391, 468 (1998); C. L. Kielkopf et al., Science 282, 111 (1998); S. E. Swalleyet al., J. Am. Chem. Soc 121, 1113 (1999); and D. M. Herman, et al., J. Am. Chem. Soc 121, 1121 (1999). With recent incorporation of hydroxypyrole groups as a recognition unit, AT and TA as well as GC and CG base pairs can now be effectively distinguished in DNA sequences by pyrrole-imidazole polyamides related to distamycin.
The pyrrole-imidazole polyamide system is the only one of the well-known minor-groove binding motifs that has been found to form the stacked-dimer recognition unit. Even netropsin, the first minor-groove binding agent to be structurally characterized in detail and a dicationic relative of the monocation distamycin (FIG. 1), does not form a dimer recognition unit. A recent crystal structure of a 2:1 netrospin-DNA complex found that the two netropsin molecules in the complex bound in the minor groove as tandem monomer units instead of the side-by-side dimer observed with distamycin. See e.g., X. Chen, et al., J. Mol. Biol. 267, 1157 (1997); X. Chen, et al, Nucleic Acids Res. 26, 5464 (1998); and X. Chen, et al, Nature Struct. Biol. 1, 169 (1994). The two charges of netropsin as well as other minor groove agents, such as the furan derivatives shown in FIG. 1, have been postulated to prevent stacked-dimer formation.
Recent evidence suggests that some monocationic cyanine dyes can form an array of stacked dimers in the DNA minor groove. See J. L. Seifert, et al., J. Am. Chem. Soc. (in press, 1999). There are, however, other monocationic minor-groove agents, such as Hoechst 33258 (see FIG. 1 and analogs, that apparently do not form dimer DNA recognition motifs. These results indicate that the electrostatic and stereochemical requirements for minor-groove recognition of DNA by dimers are very restrictive, and further suggest that stacked dimer formation by dications is unlikely.
The present invention is based on the inventors"" surprising discovery of a new class of organic dications, based on unfused-aromatic systems, that selectively recognize mixed DNA sequences (i.e., AT as well as GC base pairs) in a manner that is very sensitive to compound structure. These are the first non-peptide compounds that have mixed-sequence recognition capability and the result is particularly promising, since similar compounds readily enter cells and have generally low toxicity. See K. Hopkins et al., J. Med. Chem. 41, 3872-3878 (1998). A surprising feature of this discovery is that recognition occurs through highly cooperative dimer formation at the DNA binding site, a process that has been predicted not to occur for dications. The series of compounds provides a synthetically accessible new motif for specific recognition of DNA and control of gene expression. Such compounds accordingly find use in numerous therapies and treatments, including the treatment and prevention of opportunistic infections, cancer and other diseases of cell proliferation, and disorders of genetic origin (i.e., diseases caused by mutations of DNA and the like). Additionally, certain of the compounds of the present invention are fluorescent, and thus are useful for the detection of certain specific sequences recognized by the compounds of the invention.
Accordingly, a first aspect of the present invention is a compound of Formula I: 
wherein:
X is selected from the group consisting of O, S, and NH;
Y is CH or N;
A is CH or N;
B is selected from the group consisting of NH, O or S;
R1 is selected from the group consisting of H, loweralkyl, halogen, oxyalkyl, oxyaryl, and oxyarylakyl;
R2 and R9 are each independently selected from the group consisting of H, H2, hydroxy, lower alkyl, cycloalkyl, aryl, alkylaryl, alkoxyalkyl, hydroxycycloalkyl, alkoxycycloalkoxy, hydroxyalkyl, aminoalkyl and alkylaminoalkyl; and
R3, R4, R13 and R14 are each independently selected from the group consisting of H, lower alkyl, alkoxyalkyl, cycloalkyl, aryl, alkylaryl, hydroxyalkyl, aminoalkyl, and alkylaminoalkyl,or R3 and R4 together or R13 and R14 together represent a C2 to C10 alkyl, hydroxyalkyl, or alkylene, or R3 and R4 together or R13 and R14 together are: 
wherein n is a number from 1 to 3, and R10 is H or xe2x80x94CONHR11NR15R16, wherein R11 is lower alkyl and R15 and R16 are each independently selected from the group consisting of H and lower alkyl;
L is selected from the group consisting of: 
xe2x80x83wherein R5, R6, R7, and R8 are each individually selected from the group consisting of H, alkyl, halo, aryl, arylalkyl, aminoalkyl, aminoaryl, oxoalkyl, oxoaryl, and oxoarylalkyl; and wherein said compound of Formula I binds mixed-sequence DNA in the minor groove in a dimer formation. In a preferred embodiment of the invention, the compound of Formula I is a dication, L is: 
A is N; B is NH; X is O; Y is CH; R1, R2, R4, R5, R6, R7, R8, R9 and R14 are each H; and R3 and R13 are each H2.
A second aspect of the present invention is a method of selectively binding mixed sequence DNA comprising contacting a sample of DNA with a compound of Formula I.
A third aspect of the present invention is a method of detecting mixed DNA sequences comprising contacting a sample of DNA with a fluorescent compound of Formula I, and then observing fluorescence in the sample, the observation of fluorescence indicating that mixed DNA sequences have been bound.
A fourth aspect of the invention is a pharmaceutical formulation comprising a compound of Formula I in a pharmaceutically acceptable carrier.
Additional aspects of the invention include methods of controlling gene expression, methods of treating microbial infection, methods of treating cancer and other disorders of cell proliferation, and methods of treating disorders of genetic origin (i.e., where the disease state is caused by a gene mutation or mutations).
Other aspects of the present invention include the use of an active compound as described above for the preparation of a medicament for controlling gene expression, or medicament for treating a microbial infection, or a method of treating a disorder of genetic origin in a subject in need thereof.
The foregoing and other aspects of the present invention are explained in detail in the specification set forth below.